Diagnostic test kits employing an internal calibration system

ABSTRACT

A diagnostic test kit that employs a lateral flow assay device and a plurality of assay reagents for detecting a test analyte within a test sample is disclosed. The assay reagents include detection probes that are capable of producing a detection signal representing the presence or quantity of the test analyte in the test sample. To further enhance detection accuracy, calibration probes are also used that are capable of producing a calibration signal representing the presence or quantity of a calibration analyte. The calibration signal may be utilized to calibrate the detection signal.

BACKGROUND OF THE INVENTION

Various analytical procedures and devices are commonly employed inassays to determine the presence and/or concentration of analytes thatmay be present in a test sample. For instance, immunoassays utilizemechanisms of the immune systems, wherein antibodies are produced inresponse to the presence of antigens that are pathogenic or foreign tothe organisms. These antibodies and antigens, i.e., immunoreactants, arecapable of binding with one another, thereby causing a highly specificreaction mechanism that may be used to determine the presence orconcentration of that particular antigen in a biological sample.

There are several well-known immunoassay methods that useimmunoreactants labeled with a detectable component so that the analytemay be detected analytically. For example, “sandwich-type” assay formatstypically involve mixing the test sample with detection probesconjugated with a specific binding member (e.g., antibody) for theanalyte to form complexes between the analyte and the conjugated probes.These complexes are then allowed to contact a receptive material (e.g.,antibodies) immobilized within the detection zone. Binding occursbetween the analyte/probe conjugate complexes and the immobilizedreceptive material, thereby localizing “sandwich” complexes that aredetectable to indicate the presence of the analyte. This technique maybe used to obtain quantitative or semi-quantitative results. Someexamples of such sandwich-type assays are described in by U.S. Pat. No.4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 to Tom, et al. Analternative technique is the “competitive-type” assay. In a competitiveassay, the labeled probe is generally conjugated with a molecule that isidentical to, or an analog of, the analyte. Thus, the labeled probecompetes with the analyte of interest for the available receptivematerial. Competitive assays are typically used for detection ofanalytes such as haptens, each hapten being monovalent and capable ofbinding only one antibody molecule. Examples of competitive immunoassaydevices are described in U.S. Pat. No. 4,235,601 to Deutsch, et al.,U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 toBuechler, et al.

Many of these assays rely upon calibration to provide valid andmeaningful results, particularly for semi-quantitative and quantitativedetections. In external calibration systems, a standard curve is usuallyobtained from standard samples containing a series of a known amount ofanalyte, and the results obtained from the samples are then comparedwith the standard curve to extract the presence and/or amount of theanalyte in the sample. The external calibration method is relativelyeasy to design and simple to implement. However, it is often subject tointerference from environmental and batch-to-batch variations, and isthus unreliable.

Some internal calibration systems have thus been developed to overcomethese problems. For example, U.S. Pat. No. 5,387,503 to Selmer, et al.describes an internal calibration technique that involves mixing asample of a known volume with a predetermined amount of a calibratoranalyte and contacting the mixture with a solid support. The solidsupport contains a reagent capable of selectively binding the testanalyte in a first discrete area and a reagent capable of selectivelybinding the calibrator analyte in a second discrete area. A mixture of alabeled reagent for the test analyte and a similarly labeled reagent forthe calibrator analyte are also applied to the solid support. The amountof test analyte in the sample is determined by comparing the levels oflabeled reagent bound to the test and calibrator analytes, respectively.Unfortunately, such internal calibration techniques are not readilyincorporated into lateral flow devices, which involve heterogeneousseparation of the analyte using chromatographic methods. In addition,the requirement for pre-mixing the assay reagents is burdensome andoverly complicated, particularly for point-of-care applications in whichthe ultimate user is not a trained medical professional or technician.

As such, a need currently exists for an accurate internal calibrationsystem for lateral flow assays that is accurate, yet relativelyinexpensive, simple, and easy to use.

SUMMARY OF THE INVENTION

In accordance with one embodiment of the present invention, a diagnostictest kit is disclosed for detecting the presence or quantity of a testanalyte within a test sample. The diagnostic test kit comprises alateral flow assay device comprising a porous membrane. The porousmembrane defines a detection zone and a calibration zone. A firstreceptive material is immobilized within the detection zone and a secondreceptive material is immobilized within the calibration zone. Besidesthe lateral flow assay device, the diagnostic test kit also comprises aplurality of assay reagents, one or more of which are disposed on thelateral flow assay device. The assay reagents comprise a calibrationanalyte, detection probes, and calibration probes. The detection probesare conjugated with a first specific binding member configured topreferentially bind to the test analyte, the first receptive material,or combinations thereof. The calibration probes are conjugated with asecond specific binding member configured to preferentially bind to thecalibration analyte, the second receptive material, or combinationsthereof.

In accordance with another embodiment of the present invention, a methodfor quantitatively or semi-quantitatively detecting a test analyte usinga lateral flow device is disclosed. The device comprises a porousmembrane in communication with detection probes conjugated with a firstspecific binding member and calibration probes conjugated with a secondspecific binding member. The porous membrane also defines a detectionzone and a calibration zone. The method comprises contacting the lateralflow device with a calibration analyte; contacting the lateral flowdevice with the test sample; measuring the intensity of a detectionsignal produced at the detection zone; and measuring the intensity of acalibration signal produced at the calibration zone. The amount of thetest analyte is proportional to the intensity of the detection signal ascalibrated by the intensity of the calibration signal.

In accordance with still another embodiment of the present invention, amethod for quantitatively or semi-quantitatively detecting a testanalyte using a lateral flow device is disclosed. The device comprises aporous membrane in communication with a calibration analyte, detectionprobes conjugated with a first specific binding member, and calibrationprobes conjugated with a second specific binding member. The porousmembrane also defines a detection zone and a calibration zone. Themethod comprises contacting the lateral flow device with the testsample; measuring the intensity of a detection signal produced at thedetection zone; and measuring the intensity of a calibration signalproduced at the calibration zone. The amount of the test analyte isproportional to the intensity of the detection signal as calibrated bythe intensity of the calibration signal.

Other features and aspects of the present invention are discussed ingreater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof, directed to one of ordinary skill in the art, is setforth more particularly in the remainder of the specification, whichmakes reference to the appended figures in which:

FIG. 1 is a perspective view of one embodiment of a lateral flow assaydevice of the present invention;

FIG. 2 is a schematic illustration of one embodiment of a sandwich assayformat that may be used in accordance with the present invention;

FIG. 3 is a schematic illustration of one embodiment of an indirectassay format that may be used in accordance with the present invention;and

FIG. 4 is a schematic illustration of one embodiment of a competitiveassay format that may be used in accordance with the present invention.

Repeat use of reference characters in the present specification anddrawings is intended to represent same or analogous features or elementsof the invention.

DETAILED DESCRIPTION OF REPRESENTATIVE EMBODIMENTS Definitions

As used herein, the term “analyte” generally refers to a substance to bedetected. For instance, analytes may include antigenic substances,haptens, antibodies, and combinations thereof. Analytes include, but arenot limited to, toxins, organic compounds, proteins, peptides,microorganisms, amino acids, nucleic acids, hormones, steroids,vitamins, drugs (including those administered for therapeutic purposesas well as those administered for illicit purposes), drug intermediariesor byproducts, bacteria, virus particles and metabolites of orantibodies to any of the above substances. Specific examples of someanalytes include ferritin; creatinine kinase MB (CK-MB); digoxin;phenyloin; phenobarbitol; carbamazepine; vancomycin; gentamycin;theophylline; valproic acid; quinidine; luteinizing hormone (LH);follicle stimulating hormone (FSH); estradiol, progesterone; C-reactiveprotein; lipocalins; IgE antibodies; cytokines; vitamin B2micro-globulin; glycated hemoglobin (Gly. Hb); cortisol; digitoxin;N-acetylprocainamide (NAPA); procainamide; antibodies to rubella, suchas rubella-IgG and rubella IgM; antibodies to toxoplasmosis, such astoxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM);testosterone; salicylates; acetaminophen; hepatitis B virus surfaceantigen (HBsAg); antibodies to hepatitis B core antigen, such asanti-hepatitis B core antigen IgG and IgM (Anti-HBC); human immunedeficiency virus 1 and 2 (HIV 1 and 2); human T-cell leukemia virus 1and 2 (HTLV); hepatitis B e antigen (HBeAg); antibodies to hepatitis B eantigen (Anti-HBe); influenza virus; thyroid stimulating hormone (TSH);thyroxine (T4); total triiodothyronine (Total T3); free triiodothyronine(Free T3); carcinoembryoic antigen (CEA); lipoproteins, cholesterol, andtriglycerides; and alpha fetoprotein (AFP). Drugs of abuse andcontrolled substances include, but are not intended to be limited to,amphetamine; methamphetamine; barbiturates, such as amobarbital,secobarbital, pentobarbital, phenobarbital, and barbital;benzodiazepines, such as librium and valium; cannabinoids, such ashashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opiates,such as heroin, morphine, codeine, hydromorphone, hydrocodone,methadone, oxycodone, oxymorphone and opium; phencyclidine; andpropoxyhene. Other potential analytes may be described in U.S. Pat. No.6,436,651 to Everhart, et al. and U.S. Pat. No. 4,366,241 to Tom et al.

As used herein, the term “test sample” generally refers to a biologicalmaterial suspected of containing the analyte. The test sample may bederived from any biological source, such as a physiological fluid,including, blood, interstitial fluid, saliva, ocular lens fluid,cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, nasalfluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses,amniotic fluid, semen, and so forth. Besides physiological fluids, otherliquid samples may be used such as water, food products, and so forth,for the performance of environmental or food production assays. Inaddition, a solid material suspected of containing the analyte may beused as the test sample. The test sample may be used directly asobtained from the biological source or following a pretreatment tomodify the character of the sample. For example, such pretreatment mayinclude preparing plasma from blood, diluting viscous fluids, and soforth. Methods of pretreatment may also involve filtration,precipitation, dilution, distillation, mixing, concentration,inactivation of interfering components, the addition of reagents,lysing, etc. Moreover, it may also be beneficial to modify a solid testsample to form a liquid medium or to release the analyte.

DETAILED DESCRIPTION

Reference now will be made in detail to various embodiments of theinvention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, notlimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations may be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment, may be used on another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present inventioncovers such modifications and variations as come within the scope of theappended claims and their equivalents.

In general, the present invention is directed to a diagnostic test kitthat employs a lateral flow assay device and a plurality of assayreagents for detecting a test analyte within a test sample. The assayreagents include detection probes that are capable of producing adetection signal representing the presence or quantity of the testanalyte in the test sample. To further enhance detection accuracy,calibration probes are also used that are capable of producing acalibration signal representing the presence or quantity of acalibration analyte. The calibration signal may be utilized to calibratethe detection signal.

The calibration analyte is normally either foreign to the test sample orpresent at a constant concentration so that it is easier to distinguishbetween the test analyte and calibration analyte. Further, it is alsonormally desired that the calibration analyte exhibit a similardegradation profile (or loss of activity over time) to the test analytewith respect to conditions of pH, temperature, salt concentration, etc.In this manner, the calibration analyte will behave similarly under thesame reaction conditions and storage time so that the calibration curveof the calibration analyte will be substantially similar to thecalibration curve of the test analyte. If desired, for example, thecalibration analyte may be a member of the same protein family as thetest analyte. In one embodiment, the test analyte is C-reactive protein(“CRP”), which is a globulin that forms a precipitate with the somaticC-polysaccharide of the Streptococcus pneumoniae. CRP belongs to the“pentraxin” family of proteins, which are oligomeric plasma proteinsthat have a pentagonal cyclic symmetry with five noncovalently boundsubunits. There are two common branches of the “pentraxin” family, i.e.,“CRP-like” proteins and Serum Amyloid P (“SAP”) like proteins. Proteinsthat bind phosphocholine are considered CRP-like, while proteins thatbind carbohydrate moieties are considered SAP-like. Thus, in anembodiment in which CRP is the test analyte, the selected calibrationanalyte may be a member of the pentraxin family, and even more desirablya pentraxin protein that binds with phosphocholine. Some examples ofsuch phosphocholine-binding pentraxin proteins include, but are notlimited to, pentraxin 3 (“PTX3”), neuronal pentraxin 1, and neuronalpentraxin 2. See e.g., Arteriosclerosis, Thrombosis, and VascularBiology: Production of the Long Pentraxin PTX3 in AdvancedAtherosclerotic Plaques; Michael S. Rolph, et al.; 2002; 22:e10.However, it is not necessary that the calibration analyte is a member ofthe same family as the test analyte. In fact, many applications requirea lower level of calibration accuracy, and as such, less expensive andmore readily available calibration analytes may be employed. In oneembodiment, for example, a calibration analyte for CRP may be a proteinselected from a non-pentraxin family of proteins (e.g., dimer, trimer,etc.), such as albumin, bovine serum albumin (BSA), β-casein, or hCG,all of which are believed to have a similar degradation profile as CRP.

As stated above, detection and calibration probes are employed in thepresent invention for detecting the test analyte and calibrationanalyte, respectively. The calibration probes generally contain the sametype of detectable substance as the detection probes. Any substancecapable of producing a signal that is detectable visually or by aninstrumental device may be used as the detection or calibration probes.Suitable detectable substances may include, for instance, luminescentcompounds (e.g., fluorescent, phosphorescent, etc.); radioactivecompounds; visual compounds (e.g., colored dye or metallic substance,such as gold); liposomes or other vesicles containing signal-producingsubstances; enzymes and/or substrates, and so forth. Other suitabledetectable substances may be described in U.S. Pat. No. 5,670,381 toJou, et al. and U.S. Pat. No. 5,252,459 to Tarcha, et al., which areincorporated herein in their entirety by reference thereto for allpurposes. If the detectable substance is colored, the idealelectromagnetic radiation is light of a complementary wavelength. Forinstance, blue detection probes strongly absorb red light.

In some embodiments, the detectable substance may be a luminescentcompound that produces an optically detectable signal. For example,suitable fluorescent molecules may include, but are not limited to,fluorescein, europium chelates, phycobiliprotein, rhodamine, and theirderivatives and analogs. Other suitable fluorescent compounds aresemiconductor nanocrystals commonly referred to as “quantum dots.” Forexample, such nanocrystals may contain a core of the formula CdX,wherein X is Se, Te, S, and so forth. The nanocrystals may also bepassivated with an overlying shell of the formula YZ, wherein Y is Cd orZn, and Z is S or Se. Other examples of suitable semiconductornanocrystals may also be described in U.S. Pat. No. 6,261,779 toBarbera-Guillem, et al. and U.S. Pat. No. 6,585,939 to Dapprich, whichare incorporated herein in their entirety by reference thereto for allpurposes.

Further, suitable phosphorescent compounds may include metal complexesof one or more metals, such as ruthenium, osmium, rhenium, iridium,rhodium, platinum, indium, palladium, molybdenum, technetium, copper,iron, chromium, tungsten, zinc, and so forth. Especially preferred areruthenium, rhenium, osmium, platinum, and palladium. The metal complexmay contain one or more ligands that facilitate the solubility of thecomplex in an aqueous or nonaqueous environment. For example, somesuitable examples of ligands include, but are not limited to, pyridine;pyrazine; isonicotinamide; imidazole; bipyridine; terpyridine;phenanthroline; dipyridophenazine; porphyrin, porphine, and derivativesthereof. Such ligands may be, for instance, substituted with alkyl,substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino,hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide,sulfur-containing groups, phosphorus containing groups, and thecarboxylate ester of N-hydroxy-succinimide.

Porphyrins and porphine metal complexes possess pyrrole groups coupledtogether with methylene bridges to form cyclic structures with metalchelating inner cavities. Many of these molecules exhibit strongphosphorescence properties at room temperature in suitable solvents(e.g., water) and an oxygen-free environment. Some suitable porphyrincomplexes that are capable of exhibiting phosphorescent propertiesinclude, but are not limited to, platinum (II) coproporphyrin-I and III,palladium (II) coproporphyrin, ruthenium coproporphyrin,zinc(II)-coproporphyrin-I, derivatives thereof, and so forth. Similarly,some suitable porphine complexes that are capable of exhibitingphosphorescent properties include, but not limited to, platinum(II)tetra-meso-fluorophenylporphine and palladium(II)tetra-meso-fluorophenylporphine. Still other suitable porphyrin and/orporphine complexes are described in U.S. Pat. No. 4,614,723 to Schmidt,et al.; U.S. Pat. No. 5,464,741 to Hendrix; U.S. Pat. No. 5,518,883 toSoini; U.S. Pat. No. 5,922,537 to Ewart, et al.; U.S. Pat. No. 6,004,530to Sagner, et al.; and U.S. Pat. No. 6,582,930 to Ponomarev, et al.,which are incorporated herein in their entirety by reference thereto forall purposes.

Bipyridine metal complexes may also be utilized as phosphorescentcompounds. Some examples of suitable bipyridine complexes include, butare note limited to,bis[(4,4′-carbomethoxy)-2,2′-bipyridine]2-[3-(4-methyl-2,2′-bipyridine-4-yl)propyl]-1,3-dioxolaneruthenium (II);bis(2,2′bipyridine)[4-(butan-1-al)-4′-methyl-2,2′-bi-pyridine]ruthenium(II); bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine-4′-yl)-butyricacid]ruthenium (II); tris(2,2′bipyridine)ruthenium (II);(2,2′-bipyridine)[bis-bis(1,2-diphenylphosphino)ethylene]2-[3-(4-methyl-2,2′-bipyridine-4′-yl)propyl]-1,3-dioxolaneosmium (II);bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine)-butylamine]ruthenium(II);bis(2,2′-bipyridine)[1-bromo-4(4′-methyl-2,2′-bipyridine-4-yl)butane]ruthenium(II); bis(2,2′-bipyridine)maleimidohexanoic acid,4-methyl-2,2′-bipyridine-4′-butylamide ruthenium (II), and so forth.Still other suitable metal complexes that may exhibit phosphorescentproperties may be described in U.S. Pat. No. 6,613,583 to Richter, etal.; U.S. Pat. No. 6,468,741 to Massey, et al.; U.S. Pat. No. 6,444,423to Meade, et al.; U.S. Pat. No. 6,362,011 to Massey, et al.; U.S. Pat.No. 5,731,147 to Bard, et al.; and U.S. Pat. No. 5,591,581 to Massey, etal., which are incorporated herein in their entirety by referencethereto for all purposes.

In some cases, luminescent compounds may have a relatively long emissionlifetime and a relatively large “Stokes shift.” The term “Stokes shift”is generally defined as the displacement of spectral lines or bands ofluminescent radiation to a longer emission wavelength than theexcitation lines or bands. A relatively large Stokes shift allows theexcitation wavelength of a luminescent compound to remain far apart fromits emission wavelengths and is desirable because a large differencebetween excitation and emission wavelengths makes it easier to eliminatethe reflected excitation radiation from the emitted signal. Further, alarge Stokes shift also minimizes interference from luminescentmolecules in the sample and/or light scattering due to proteins orcolloids, which are present with some body fluids (e.g., blood). Inaddition, a large Stokes shift also minimizes the requirement forexpensive, high-precision filters to eliminate background interference.For example, in some embodiments, the luminescent compounds have aStokes shift of greater than about 50 nanometers, in some embodimentsgreater than about 100 nanometers, and in some embodiments, from about100 to about 350 nanometers.

For example, exemplary fluorescent compounds having a large Stokes shiftinclude lanthanide chelates of samarium (Sm (III)), dysprosium (Dy(III)), europium (Eu (III)), and terbium (Tb (III)). Such chelates mayexhibit strongly red-shifted, narrow-band, long-lived emission afterexcitation of the chelate at substantially shorter wavelengths.Typically, the chelate possesses a strong ultraviolet excitation banddue to a chromophore located close to the lanthanide in the molecule.Subsequent to excitation by the chromophore, the excitation energy maybe transferred from the excited chromophore to the lanthanide. This isfollowed by a fluorescence emission characteristic of the lanthanide.Europium chelates, for instance, have Stokes shifts of about 250 toabout 350 nanometers, as compared to only about 28 nanometers forfluorescein. Also, the fluorescence of europium chelates is long-lived,with lifetimes of about 100 to about 1000 microseconds, as compared toabout 1 to about 100 nanoseconds for other fluorescent labels. Inaddition, these chelates have a narrow emission spectra, typicallyhaving bandwidths less than about 10 nanometers at about 50% emission.One suitable europium chelate is N-(p-isothiocyanatobenzyl)-diethylenetriamine tetraacetic acid-Eu⁺³.

In addition, lanthanide chelates that are inert, stable, andintrinsically fluorescent in aqueous solutions or suspensions may alsobe used in the present invention to negate the need for micelle-formingreagents, which are often used to protect chelates having limitedsolubility and quenching problems in aqueous solutions or suspensions.One example of such a chelate is4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6-bis([N,N-bis(carboxymethyl)amino]methyl)-pyridine[Ref: Lovgren, T., et al.; Clin. Chem. 42, 1196-1201 (1996)]. Severallanthanide chelates also show exceptionally high signal-to-noise ratios.For example, one such chelate is a tetradentate β-diketonate-europiumchelate [Ref: Yuan, J. and Matsumoto, K.; Anal. Chem. 70, 596-601(1998)]. In addition to the fluorescent labels described above, otherlabels that are suitable for use in the present invention may bedescribed in U.S. Pat. No. 6,030,840 to Mullinax, et al.; U.S. Pat. No.5,585,279 to Davidson; U.S. Pat. No. 5,573,909 to Singer, et al.; U.S.Pat. No. 6,242,268 to Wieder, et al.; and U.S. Pat. No. 5,637,509 toHemmila, et al., which are incorporated herein in their entirety byreference thereto for all purposes.

Detectable substances, such as described above, may be used alone or inconjunction with a particle (sometimes referred to as “beads” or“microbeads”). For instance, naturally occurring particles, such asnuclei, mycoplasma, plasmids, plastids, mammalian cells (e.g.,erythrocyte ghosts), unicellular microorganisms (e.g., bacteria),polysaccharides (e.g., agarose), etc., may be used. Further, syntheticparticles may also be utilized. For example, in one embodiment, latexmicroparticles that are labeled with a fluorescent or colored dye areutilized. Although any synthetic particle may be used in the presentinvention, the particles are typically formed from polystyrene,butadiene styrenes, styreneacrylic-vinyl terpolymer,polymethylmethacrylate, polyethylmethacrylate, styrene-maleic anhydridecopolymer, polyvinyl acetate, polyvinylpyridine, polydivinylbenzene,polybutyleneterephthalate, acrylonitrile, vinylchloride-acrylates, andso forth, or an aldehyde, carboxyl, amino, hydroxyl, or hydrazidederivative thereof. Other suitable particles may be described in U.S.Pat. No. 5,670,381 to Jou, et al. and U.S. Pat. No. 5,252,459 to Tarcha,et al. Commercially available examples of suitable fluorescent particlesinclude fluorescent carboxylated microspheres sold by Molecular Probes,Inc. under the trade names “FluoSphere” (Red 580/605) and“TransfluoSphere” (543/620), as well as “Texas Red” and 5- and6-carboxytetramethylrhodamine, which are also sold by Molecular Probes,Inc. In addition, commercially available examples of suitable colored,latex microparticles include carboxylated latex beads sold by Bang'sLaboratory, Inc. Metallic particles (e.g., gold particles) may also beutilized in the present invention.

When utilized, the shape of the particles may generally vary. In oneparticular embodiment, for instance, the particles are spherical inshape. However, it should be understood that other shapes are alsocontemplated by the present invention, such as plates, rods, discs,bars, tubes, irregular shapes, etc. In addition, the size of theparticles may also vary. For instance, the average size (e.g., diameter)of the particles may range from about 0.1 nanometers to about 1,000microns, in some embodiments, from about 0.1 nanometers to about 100microns, and in some embodiments, from about 1 nanometer to about 10microns.

It is generally desired to modify the detection and calibration probesin some manner so that they are more readily able to bind to arespective analyte or receptive material. In such instances, the probesmay be modified with certain specific binding members that are adheredthereto to form conjugated probes. Specific binding members generallyrefer to a member of a specific binding pair, i.e., two differentmolecules where one of the molecules chemically and/or physically bindsto the second molecule. The selection of the specific binding membergenerally depends on the test analyte of interest and on thecorresponding calibration analyte. To ensure independent assayperformance, it is normally desired that the detection probes areconjugated with a member of a different specific binding pair than thecalibration probes. In this manner, the conjugated calibration probeswill preferentially bind with the calibration analyte (sandwich andindirect assay formats) or with a specific binding member for thecalibration analyte (competitive assay formats). The conjugatedcalibration probes will not, however, generally bind with the testanalyte or with a specific binding member for the test analyte. As such,assays may be simultaneously performed for the test analyte and thecalibration analyte without fear of substantial cross-reaction, therebyallowing the calibration analyte assay to be used in calibrating thetest antigen. Also, similar to the relationship between the calibrationanalyte and the test analyte, it is normally desired that the specificbinding members exhibit a similar degradation profile with respect toconditions of pH, temperature, salt concentration, storage time, etc.

Some examples of suitable immunoreactive specific binding members thatmay be used in the present invention include, but are not limited to,antigens, haptens, aptamers, antibodies (primary or secondary), andcomplexes thereof, including those formed by recombinant DNA methods orpeptide synthesis. An antibody may be a monoclonal or polyclonalantibody, a recombinant protein or a mixture(s) or fragment(s) thereof,as well as a mixture of an antibody and other specific binding members.The details of the preparation of such antibodies and their suitabilityfor use as specific binding members are well known to those skilled inthe art. Other common specific binding pairs include but are not limitedto, biotin and avidin (or derivatives thereof), biotin and streptavidin,carbohydrates and lectins, complementary nucleotide sequences (includingprobe and capture nucleic acid sequences used in DNA hybridizationassays to detect a target nucleic acid sequence), complementary peptidesequences including those formed by recombinant methods, effector andreceptor molecules, hormone and hormone binding protein, enzymecofactors and enzymes, enzyme inhibitors and enzymes, and so forth.Furthermore, specific binding pairs may include members that are analogsof the original specific binding member. For example, a derivative orfragment of the analyte, i.e., an analyte-analog, may be used so long asit has at least one epitope in common with the analyte.

The specific binding members may generally be attached to the probesusing any of a variety of well-known techniques. For instance, covalentattachment of the specific binding members to the detection probes(e.g., particles) may be accomplished using carboxylic, amino, aldehyde,bromoacetyl, iodoacetyl, thiol, epoxy and other reactive or linkingfunctional groups, as well as residual free radicals and radicalcations, through which a protein coupling reaction may be accomplished.A surface functional group may also be incorporated as a functionalizedco-monomer because the surface of the probe may contain a relativelyhigh surface concentration of polar groups. In addition, although probesare often functionalized after synthesis, such as with poly(thiophenol),the probes may be capable of direct covalent linking with a proteinwithout the need for further modification. For example, in oneembodiment, the first step of conjugation is activation of carboxylicgroups on the probe surface using carbodiimide. In the second step, theactivated carboxylic acid groups are reacted with an amino group of anantibody to form an amide bond. The activation and/or antibody couplingmay occur in a buffer, such as phosphate-buffered saline (PBS) (e.g., pHof 7.2) or 2-(N-morpholino) ethane sulfonic acid (MES) (e.g., pH of5.3). The resulting probes may then be contacted with ethanolamine, forinstance, to block any remaining activated sites. Overall, this processforms a conjugated probe, where the antibody is covalently attached tothe probe. Besides covalent bonding, other attachment techniques, suchas physical adsorption, may also be utilized in the present invention.

Regardless of the manner in which they are formed, the detection probesand calibration probes are typically disposed on the assay device priorto application of the test sample. The pre-application of the probes tothe assay device provides a variety of benefits. For example,pre-application eliminates the need for a subsequent user to handle andmix the reagents with the test sample or a diluent. This is particularlyuseful in point-of-care applications where the user is not generally atrained lab technician or medical professional. In some embodiments, forexample, the detection and calibration probes are disposed downstreamfrom the point where the test sample is to be applied. In this manner,the test sample is capable of mixing with and optionally re-suspendingthe probes upon application. Alternatively, the probes may be positionedupstream from the point of application of the test sample. For instance,a diluent may be employed to re-suspend the probes for performing theassay. Similarly, the calibration analyte may also be disposed on theassay device prior to application of the test sample. Although theparticular location may vary, it is generally desired that thecalibration analyte is applied upstream from the detection probes andcalibration probes. In this manner, the calibration analyte is able toreadily mix with the test sample before contacting the calibrationprobes, thereby enhancing binding therebetween. The calibration analytemay also be mixed with the test sample prior to application to the assaydevice. As such, the calibration analyte is subjected to substantiallythe same conditions as the test analyte prior to performance of theassay. This may further optimize calibration accuracy.

Referring now to FIG. 1, one embodiment of a lateral flow assay device20 that may be employed in conjunction with the internal calibrationsystem of the present invention will now be described in more detail. Asshown, the device 20 contains a porous membrane 23 optionally supportedby a rigid material 21. In general, the porous membrane 23 may be madefrom any of a variety of materials through which the test sample iscapable of passing. For example, the materials used to form the porousmembrane 23 may include, but are not limited to, natural, synthetic, ornaturally occurring materials that are synthetically modified, such aspolysaccharides (e.g., cellulose materials such as paper and cellulosederivatives, such as cellulose acetate and nitrocellulose); polyethersulfone; polyethylene; nylon; polyvinylidene fluoride (PVDF); polyester;polypropylene; silica; inorganic materials, such as deactivated alumina,diatomaceous earth, MgSO₄, or other inorganic finely divided materialuniformly dispersed in a porous polymer matrix, with polymers such asvinyl chloride, vinyl chloride-propylene copolymer, and vinylchloride-vinyl acetate copolymer; cloth, both naturally occurring (e.g.,cotton) and synthetic (e.g., nylon or rayon); porous gels, such assilica gel, agarose, dextran, and gelatin; polymeric films, such aspolyacrylamide; and so forth. In one particular embodiment, the porousmembrane 23 is formed from nitrocellulose and/or polyether sulfonematerials. It should be understood that the term “nitrocellulose” refersto nitric acid esters of cellulose, which may be nitrocellulose alone,or a mixed ester of nitric acid and other acids, such as aliphaticcarboxylic acids having from 1 to 7 carbon atoms.

The size and shape of the porous membrane 23 may generally vary as isreadily recognized by those skilled in the art. For instance, a porousmembrane strip may have a length of from about 10 to about 100millimeters, in some embodiments from about 20 to about 80 millimeters,and in some embodiments, from about 40 to about 60 millimeters. Thewidth of the membrane strip may also range from about 0.5 to about 20millimeters, in some embodiments from about 1 to about 15 millimeters,and in some embodiments, from about 2 to about 10 millimeters. Likewise,the thickness of the membrane strip is generally small enough to allowtransmission-based detection. For example, the membrane strip may have athickness less than about 500 micrometers, in some embodiments less thanabout 250 micrometers, and in some embodiments, less than about 150micrometers.

As stated above, the support 21 carries the porous membrane 23. Forexample, the support 21 may be positioned directly adjacent to theporous membrane 23 as shown in FIG. 1, or one or more intervening layersmay be positioned between the porous membrane 23 and the support 21.Regardless, the support 21 may generally be formed from any materialable to carry the porous membrane 23. The support 21 may be formed froma material that is transmissive to light, such as transparent oroptically diffuse (e.g., transluscent) materials. Also, it is generallydesired that the support 21 is liquid-impermeable so that fluid flowingthrough the membrane 23 does not leak through the support 21. Examplesof suitable materials for the support include, but are not limited to,glass; polymeric materials, such as polystyrene, polypropylene,polyester (e.g., Mylar® film), polybutadiene, polyvinylchloride,polyamide, polycarbonate, epoxides, methacrylates, and polymelamine; andso forth. To provide a sufficient structural backing for the porousmembrane 23, the support 21 is generally selected to have a certainminimum thickness. Likewise, the thickness of the support 21 istypically not so large as to adversely affect its optical properties.Thus, for example, the support 21 may have a thickness that ranges fromabout 100 to about 5,000 micrometers, in some embodiments from about 150to about 2,000 micrometers, and in some embodiments, from about 250 toabout 1,000 micrometers. For instance, one suitable membrane striphaving a thickness of about 125 micrometers may be obtained fromMillipore Corp. of Bedford, Mass. under the name “SHF180UB25.”

As is well known the art, the porous membrane 23 may be cast onto thesupport 21, wherein the resulting laminate may be die-cut to the desiredsize and shape. Alternatively, the porous membrane 23 may simply belaminated to the support 21 with, for example, an adhesive. In someembodiments, a nitrocellulose or nylon porous membrane is adhered to aMylar® film. An adhesive is used to bind the porous membrane to theMylar® film, such as a pressure-sensitive adhesive. Laminate structuresof this type are believed to be commercially available from MilliporeCorp. of Bedford, Mass. Still other examples of suitable laminate assaydevice structures are described in U.S. Pat. No. 5,075,077 to Durley,III, et al., which is incorporated herein in its entirety by referencethereto for all purposes.

The device 20 may also contain an absorbent pad 28. The absorbent pad 28generally receives fluid that has migrated through the entire porousmembrane 23. As is well known in the art, the absorbent pad 28 mayassist in promoting capillary action and fluid flow through the membrane23.

To initiate the detection of an analyte within the test sample, a usermay directly apply the test sample to a portion of the porous membrane23 through which it may then travel in the direction illustrated byarrow “L” in FIG. 1. Alternatively, the test sample may first be appliedto a sample pad 24 that is in fluid communication with the porousmembrane 23. Some suitable materials that may be used to form the samplepad 24 include, but are not limited to, nitrocellulose, cellulose,porous polyethylene pads, and glass fiber filter paper. If desired, thesample pad 24 may also contain one or more assay pretreatment reagents,either diffusively or non-diffusively attached thereto. For example, inone embodiment, the calibration analyte may be disposed on the samplepad 24 so that it contacts the test sample upon application thereto.

In the illustrated embodiment, the test sample travels from the samplepad 24 to a conjugate pad 22 that is placed in communication with oneend of the sample pad 24. The conjugate pad 22 is formed from a materialthrough which the test sample is capable of passing. For example, in oneembodiment, the conjugate pad 22 is formed from glass fibers. Althoughonly one conjugate pad 22 is shown, it should be understood thatmultiple conjugate pads may also be used in the present invention. Inone particular embodiment of the present invention, the detection andcalibration probes (not shown) are applied to the conjugate pad 22.After application, the probes are then dried to inhibit migrationtherefrom. The conjugate pad 22 provides a matrix for the deposition ofthe probes so that they are free to migrate when rehydrated. Morespecifically, when a liquid test sample contacts the probes, they arerehydrated and become re-suspended and/or re-solubilized. Of course, itshould be understood that the probes may be applied to various otherlocations of the assay device 20 as well, such as directly to themembrane 23, so long as they are capable of being rehydrated by the testsample upon contact therewith.

Referring again to FIG. 1, the porous membrane 23 also defines variouszones configured to perform the assay. For instance, the porous membrane23 defines a detection zone 31 that contains a first receptive materialthat is capable of binding to the conjugated detection probes (orcomplexes thereof) that pass through the length of the membrane 23. Thefirst receptive material is immobilized on the porous membrane 23 andmay be selected from the same materials as the specific binding membersdescribed above, including, for instance, antigens; haptens;antibody-binding proteins, such as protein A, protein G, or protein A/G;neutravidin (a deglysolated avidin derivative), avidin (a highlycationic 66,000-dalton glycoprotein), streptavidin (a nonglycosylated52,800-dalton protein), or captavidin (a nitrated avidin derivative);primary or secondary antibodies, and derivatives or fragments thereof.In some embodiments, the first receptive material is an antibodyspecific to an antigen within the test sample. In sandwich assayformats, for example, the first receptive material may serve as astationary binding site for complexes formed between the analyte and theconjugated detection probes. Specifically, analytes, such as antibodies,antigens, etc., typically have two or more binding sites (e.g.,epitopes). Upon reaching the detection zone 31, one of these bindingsites is occupied by the specific binding member of the conjugatedprobe. However, the free binding site of the analyte may bind to theimmobilized first receptive material. Upon being bound to theimmobilized receptive material, the complexed probes form a new ternarysandwich complex.

The assay device 20 also includes a calibration zone 32. In thisembodiment, the calibration zone 32 is formed on the porous membrane 23and is positioned downstream from the detection zone 31. Alternatively,however, the calibration zone 32 may also be positioned upstream fromthe detection zone 31. The calibration zone 32 is provided with a secondreceptive material that is capable of binding to the conjugatedcalibration probes (or complexes thereof) that pass through the lengthof the membrane 23. The second receptive material may be a member of thespecific binding pair used for conjugating the calibration probes. Inthis manner, the second receptive material preferentially binds to thecalibration probes (or complexes thereof). For example, when thecalibration analyte is an antigen, the second receptive material may bean antibody (e.g., sandwich or competitive assay formats) or an antigen(e.g., indirect assay format). Also, as discussed above, it is normallydesired that the first and second receptive materials exhibit a similardegradation profile with respect to conditions of pH, temperature, saltconcentration, storage time, etc.

The detection zone 31 and calibration zone 32 may each provide anynumber of distinct detection regions so that a user may better determinethe concentration of the analyte within the test sample. Each region maycontain the same receptive materials, or may contain different receptivematerials. For example, the zones may include two or more distinctregions (e.g., lines, dots, etc.). The regions may be disposed in theform of lines in a direction that is substantially perpendicular to theflow of the test sample through the assay device 20. Likewise, in someembodiments, the regions may be disposed in the form of lines in adirection that is substantially parallel to the flow of the test samplethrough the assay device 20.

When it is desired to semi-quantitatively or quantitatively determinethe concentration of the analyte within the test sample, the intensityof the detection signal “I_(s)” produced at the detection zone 31 may becompared with the intensity of the calibration signal “I_(c)” producedat the calibration zone 32. For example, in some embodiments (e.g.,sandwich assay formats), the quantity of analyte is directlyproportional to the ratio of I_(s) to I_(c). In other embodiments (e.g.,competitive and indirect assay formats), the quantity of analyte inexcess of the predefined base quantity is inversely proportional to theratio of I_(s) to I_(c). Based upon the range in which the ratio falls,the general concentration range for the analyte may be determined. Ifdesired, the ratio of I_(s) to I_(c) may be plotted versus the analyteconcentration for a range of known analyte concentrations to generate acalibration curve. To determine the quantity of analyte in an unknowntest sample that is in excess of a predefined base quantity, the signalratio may then be converted to analyte concentration according to thecalibration curve. It should be noted that alternative mathematicalrelationships between I_(s) and I_(c) may be plotted versus the analyteconcentration to generate the calibration curve. For example, in oneembodiment, the value of I_(s)/(I_(s)+I_(c)) may be plotted versusanalyte concentration to generate the calibration curve. Regardless,calibration and sample testing may be conducted under approximately thesame conditions at the same time, thus providing reliable quantitativeor semi-quantitative results, with increased sensitivity.

If desired, an optical reader may be used in some embodiments to measurethe intensity of the probes at the detection zone 31 and/or calibrationzone 32. The actual configuration and structure of the optical readermay generally vary as is readily understood by those skilled in the art.For example, optical detection techniques that may be utilized include,but are not limited to, luminescence (e.g., fluorescence,phosphorescence, etc.), absorbance (e.g., fluorescent ornon-fluorescent), diffraction, etc. One suitable reflectancespectrophotometer is described, for instance, in U.S. Patent App. Pub.No. 2003/0119202 to Kaylor, et al., which is incorporated herein in itsentirety by reference thereto for all purposes. In another embodiment, areflectance-mode spectrofluorometer may be used to detect the presenceof probes that exhibit fluorescence. Suitable spectrofluorometers andrelated detection techniques are described, for instance, in U.S. PatentApp. Pub. No. 2004/0043502 to Song, et al., which is incorporated hereinin its entirety by reference thereto for all purposes. Likewise, atransmission-mode detection system may also be used to detect thepresence of detection probes.

Typically, the optical reader contains an illumination source that iscapable of emitting electromagnetic radiation and a detector that iscapable of registering a signal (e.g., transmitted or reflected light,emitted fluorescence or phosphorescence, etc.). The illumination sourcemay be any device known in the art that is capable of providingelectromagnetic radiation, such as light in the visible or near-visiblerange (e.g., infrared or ultraviolet light). For example, suitableillumination sources that may be used in the present invention include,but are not limited to, light emitting diodes (LED), flashlamps,cold-cathode fluorescent lamps, electroluminescent lamps, and so forth.The illumination may be multiplexed and/or collimated. In some cases,the illumination may be pulsed to reduce any background interference.Further, illumination may be continuous or may combine continuous wave(CW) and pulsed illumination where multiple illumination beams aremultiplexed (e.g., a pulsed beam is multiplexed with a CW beam),permitting signal discrimination between a signal induced by the CWsource and a signal induced by the pulsed source. For example, in someembodiments, LEDs (e.g., aluminum gallium arsenide red diodes, galliumphosphide green diodes, gallium arsenide phosphide green diodes, orindium gallium nitride violet/blue/ultraviolet (UV) diodes) are used asthe pulsed illumination source. One commercially available example of asuitable UV LED excitation diode suitable for use in the presentinvention is Model NSHU55OE (Nichia Corporation), which emits 750 to1000 microwatts of optical power at a forward current of 10 milliamps(3.5-3.9 volts) into a beam with a full-width at half maximum of 10degrees, a peak wavelength of 370-375 nanometers, and a spectralhalf-width of 12 nanometers.

In some cases, the illumination source may provide diffuse illuminationto the assay device. For example, an array of multiple point lightsources (e.g., LEDs) may simply be employed to provide relativelydiffuse illumination. Another particularly desired illumination sourcethat is capable of providing diffuse illumination in a relativelyinexpensive manner is an electroluminescent (EL) device. An EL device isgenerally a capacitor structure that utilizes a luminescent material(e.g., phosphor particles) sandwiched between electrodes, at least oneof which is transparent to allow light to escape. Application of avoltage across the electrodes generates a changing electric field withinthe luminescent material that causes it to emit light.

The detector may generally be any device known in the art that iscapable of sensing a signal. For instance, the detector may be anelectronic imaging detector that is configured for spatialdiscrimination. Some examples of such electronic imaging sensors includehigh speed, linear charge-coupled devices (CCD), charge-injectiondevices (CID), complementary-metal-oxide-semiconductor (CMOS) devices,and so forth. Such image detectors, for instance, are generallytwo-dimensional arrays of electronic light sensors, although linearimaging detectors (e.g., linear CCD detectors) that include a singleline of detector pixels or light sensors, such as, for example, thoseused for scanning images, may also be used. Each array includes a set ofknown, unique positions that may be referred to as “addresses.” Eachaddress in an image detector is occupied by a sensor that covers an area(e.g., an area typically shaped as a box or a rectangle). This area isgenerally referred to as a “pixel” or pixel area. A detector pixel, forinstance, may be a CCD, CID, or a CMOS sensor, or any other device orsensor that detects or measures light. The size of detector pixels mayvary widely, and may in some cases have a diameter or length as low as0.2 micrometers.

In other embodiments, the detector may be a light sensor that lacksspatial discrimination capabilities. For instance, examples of suchlight sensors may include photomultiplier devices, photodiodes, such asavalanche photodiodes or silicon photodiodes, and so forth. Siliconphotodiodes are sometimes advantageous in that they are inexpensive,sensitive, capable of high-speed operation (short risetime/highbandwidth), and easily integrated into most other semiconductortechnology and monolithic circuitry. In addition, silicon photodiodesare physically small, which enables them to be readily incorporated intoa system for use with a membrane-based device. If silicon photodiodesare used, then the wavelength range of the emitted signal may be withintheir range of sensitivity, which is 400 to 1100 nanometers.

Detection and calibration may be performed automatically and/or manuallyin accordance with the present invention. For example, a microprocessormay optionally be employed to convert signal intensities from a detectorto a result that quantitatively or semi-quantitatively indicates theconcentration of the analyte. The microprocessor may include memorycapability to allow the user to recall the last several results. Thoseskilled in the art will appreciate that any suitable computer-readablememory devices, such as RAM, ROM, EPROM, EEPROM, flash memory cards,digital video disks, Bernoulli cartridges, and so forth, may be used. Ifdesired, the results may be conveyed to a user using a liquid crystal(LCD) or LED display.

Referring to FIG. 2, one embodiment of a method for detecting thepresence of a test antigen A (e.g., CRP) using a sandwich assay formatwill now be described in more detail. Initially, a calibration antigenA* (e.g., PTX3) is pre-applied to the sample pad 24, and conjugateddetection probes 41 and conjugated calibration probes 43 are pre-appliedto the conjugate pad 22. In one embodiment, for example, the detectionprobes 41 are dyed particles conjugated with an antibody for CRP (e.g.,CRP IgG₁) and the calibration probes 43 are dyed particles conjugatedwith an antibody for PTX3 that does not cross-react with other pentraxinfamily members (e.g., rat anti-body PTX3 (clone MNB4)). To initiate theassay, a test sample containing the test antigen A is applied to thesample pad 22 where it mixes with the calibration antigen A*. The testantigen A and calibration antigen A* travel in a direction “L” to theconjugate pad 22, where they mix with the detection probes 41 andcalibration probes 43. The test antigen A binds with the detectionprobes 41 to form analyte/probe complexes 49, and the calibrationantigen A* binds with the calibration probes 43 to form analyte/probecomplexes 50. The complexes 49 and 50 then travel to the detection zone31, within which is immobilized a first receptive material 51. Forexample, the first receptive material may be an antibody for CRP that isdifferent than the antibody of the conjugated detection probes (e.g.,anti-CRP IgG₂) or an antibody for CRP that is identical to the antibodyconjugated to the detection probes. The complexes 49 bind to theavailable binding sites on the immobilized receptive material 51. Theremaining complexes 50 then travel to the calibration zone 32, withinwhich is immobilized a second receptive material 53. For example, thesecond receptive material may be an antibody for PTX3 that is the sameor different than the antibody of the conjugated calibration probes(e.g., a different clone of rat anti-body PTX3). The complexes 50 bindto the available binding sites on the immobilized receptive material 53.The intensity of the signals produced by the detection probes 41captured at the detection zone 31 and the calibration probes 43 at thecalibration zone 32 may then be measured. Referring to FIG. 3, anotherembodiment of a method for detecting the presence of a test antigen Ausing an indirect assay format will now be described in more detail. Inthis embodiment, the detection probes 41 are conjugated with an antibodyfor the test antigen A, and the calibration probes 43 are conjugatedwith an antibody for the calibration antigen A*. In addition, the firstreceptive material 51 is similar in nature to the test antigen A, andthe second receptive material 53 is similar in nature to the calibrationantigen A*. In this manner, the test antigen A and the first receptivematerial 51 compete for the available binding sites on the conjugateddetection probes 41, and the calibration antigen A* and the secondreceptive material 53 compete for the available binding sites on theconjugated calibration probes 43.

Also, referring to FIG. 4, still another embodiment of a method fordetecting the presence of a test antigen A using a competitive assayformat will now be described in more detail. In this embodiment, thedetection probes 41 are conjugated with an antigen that is similar innature to the test antigen A, and the calibration probes 43 areconjugated with an antigen that is similar in nature to the calibrationantigen A*. In addition, the first receptive material 51 is an antibodyfor the test antigen A, and the second receptive material 53 is anantibody for the calibration antigen A*. In this manner, the testantigen A and the conjugated detection probes 41 compete for theavailable binding sites on the first receptive material 51, and thecalibration antigen A* and the conjugated calibration probes 43 competefor the available binding sites on the second receptive material 53.

Although various embodiments of device configurations have beendescribed above, it should be understood, that a device of the presentinvention may generally have any configuration desired, and need notcontain all of the components described above. Various other deviceconfigurations, for instance, are described in U.S. Pat. No. 5,395,754to Lambotte, et al.; U.S. Pat. No. 5,670,381 to Jou, et al.; and U.S.Pat. No. 6,194,220 to Malick, et al., which are incorporated herein intheir entirety by reference thereto for all purposes.

The present invention may be better understood with reference to thefollowing examples.

EXAMPLE 1

The ability to form a lateral flow assay device in accordance with thepresent invention was demonstrated. A nitrocellulose porous membrane (HF120 from Millipore, Inc.) having a length of approximately 30centimeters was laminated onto supporting cards. Monoclonal antibody forC-reactive protein was immobilized on the porous membrane to form adetection zone. The antibody was obtained from BiosPacific, Inc.(Catalog #A58040136P) and had a concentration of 1 milligram permilliliter. Monoclonal antibody for β-HCG (1 milligram per milliliter)was also immobilized on the porous membrane to form a calibration zone.A cellulose wicking pad (Millipore Co.) was laminated with one end(closer to the calibration zone) of the membrane. The membrane sampleswere then dried for 1 hour at a temperature of 37° C.

A particle suspension was formed by mixing 36 microliters of goldparticles conjugated with anti-CRP monoclonal antibody (BiosPacific,Inc., Catalog #A58110228P), 100 microliters of gold particles conjugatedwith anti α-hCG monoclonal antibody, 500 milliliters of sucrose in water(20%) and 1200 microliters of water. The gold particles conjugated withanti CRP monoclonal antibody had a particle size of 40 nanometers and anoptical density of 50.2, and were obtained from British BiocellInternational. The gold particles conjugated with anti α-hCG monoclonalantibody had a particle size of 40 nanometers and an optical density of10.1, and were obtained from British Biocell International, Inc. Theparticle suspension was then loaded onto a 30-centimeter long glassfiber conjugate pad (Millipore Co.). The glass fiber pad was then driedat 37° C. for 2 hours to form a conjugate pad. The conjugate pad wasthen laminated onto the other end (closer to the detection zone) of theporous membrane. A cellulose wicking pad (Millipore Co.) sample pad wasfurther laminated onto the conjugate pad. The laminated full card wasthen cut into 4-millimeter wide lateral flow assay devices.

EXAMPLE 2

The ability to detect CRP in accordance with the present invention wasdemonstrated. Five lateral flow devices (Samples 1-5) were made asdescribed in Example 1. A mixing solution containing 60 microliters ofCRP (90 nanograms per milliliter) and 60 microliters of β-hCG (50nanograms per milliliter) was applied to the sample pad of each of fivelateral flow devices. After 30 minutes of development at roomtemperature, the color density of the detection zone and calibrationzone was measured using a reflectance spectrophotometer. The colorintensity for each zone is listed below in Table 1.

TABLE 1 Color Intensity Data Detection Zone Detection Zone SampleIntensity (I_(d)) Intensity (I_(c)) I_(d)/I_(c) 1 88.285 95.279 0.926592 93.554 98.322 0.95115 3 91.850 98.873 0.92897 4 94.286 101.544 0.928525 92.580 99.990 0.92589

The mean, standard deviation, and % CV (coefficient of variance, orratio of standard deviation to mean) for I_(d) was 92.111, 2.33127, and2.531, respectively. On the other hand, the mean, standard deviation,and % CV for I_(d)/I_(c) was 0.9323, 0.01081, and 1.1159, respectively.Thus, as indicated, the standard deviation and % CV were much lower forthe calibrated signal intensity than the uncalibrated signal intensity.

EXAMPLE 3

The ability to detect CRP in accordance with the present invention wasdemonstrated. Fifteen lateral flow devices (Samples 1-15) were made asdescribed in Example 1 and divided into three groups. The first groupwas stored at room temperature, the second group was heated at 65° C.for 40 minutes, and the third group was heated at 65° C. for 210minutes. A mixing solution containing 60 microliters of CRP (100nanograms per milliliter) and 60 microliters of β-hCG (50 nanograms permilliliter) in tris buffer with 1% Tween® 20 (ICI Americas) was appliedto the sample pad of each of fifteen lateral flow devices. After 30minutes of development at room temperature, the color density of thedetection zone and calibration zone was measured using a reflectancespectrophotometer. The color intensities for each group are listed belowin Tables 2-4.

TABLE 2 Color Intensity Data for First Group Detection Zone DetectionZone Sample Intensity (I_(d)) Intensity (I_(c)) I_(d)/I_(c) 1 94.30399.106 0.95154 2 93.980 97.933 0.95964 3 94.719 97.618 0.97030 4 90.33295.088 0.94998 5 93.343 96.032 0.97200

The mean, standard deviation, and % CV for I_(d) was 93.3354, 1.75261,and 1.878, respectively. On the other hand, the mean, standarddeviation, and % CV for I_(d)/I_(c) was 0.96069, 0.01024, and 1.066,respectively.

TABLE 3 Color Intensity Data for Second Group Detection Zone DetectionZone Sample Intensity (I_(d)) Intensity (I_(c)) I_(d)/I_(c) 1 90.911094.189 0.96520 2 90.4130 94.266 0.95913 3 90.4860 93.614 0.96659 490.4399 96.666 0.97655 5 85.2780 88.978 0.95842

The mean, standard deviation, and % CV for I_(d) was 90.2974, 3.25741,and 3.607, respectively. On the other hand, the mean, standarddeviation, and % CV for I_(d)/I_(c) was 0.96517, 0.00732, and 0.758,respectively.

TABLE 4 Color Intensity Data for Third Group Detection Zone DetectionZone Sample Intensity (I_(d)) Intensity (I_(c)) I_(d)/I_(c) 1 89.27093.359 0.95620 2 87.707 91.698 0.95968 3 90.181 92.496 0.97497 4 84.39689.233 0.94579 5 87.677 91.019 0.96328

The mean, standard deviation, and % CV for I_(d) was 87.842, 2.204, and2.509, respectively. On the other hand, the mean, standard deviation,and % CV for I_(d)/I_(c) was 0.95935, 0.01074, and 1.12, respectively.

EXAMPLE 4

The ability to form a lateral flow assay device in accordance with thepresent invention was demonstrated. A nitrocellulose porous membrane (HF120 from Millipore, Inc.) having a length of approximately 30centimeters was laminated onto supporting cards. Monoclonal antibody forC-reactive protein was immobilized on the porous membrane to form adetection zone. The antibody was obtained from BiosPacific, Inc.(Catalog #A58040136P) and had a concentration of 1 milligram permilliliter. Monoclonal antibody for β-HCG (1 milligram per milliliter)was also immobilized on the porous membrane to form a calibration zone.A cellulose wicking pad (Millipore Co.) was laminated with one end(closer to the calibration zone) of the membrane. The membrane sampleswere then dried for 1 hour at a temperature of 37° C.

A particle suspension was formed by mixing 36 microliters of goldparticles conjugated with anti-CRP monoclonal antibody (BiosPacific,Inc., Catalog #A58110228P), 100 microliters of gold particles conjugatedwith anti α-hCG monoclonal antibody, 500 milliliters of sucrose in water(20%) and 1400 microliters of water. The gold particles conjugated withanti CRP monoclonal antibody had a particle size of 40 nanometers and anoptical density of 50.2, and were obtained from British BiocellInternational. The gold particles conjugated with anti a-hCG monoclonalantibody had a particle size of 40 nanometers and an optical density of10.1, and were obtained from British Biocell International. The particlesuspension was then loaded onto a 30-centimeter long glass fiberconjugate pad (Millipore Co.). The glass fiber pad was then dried at 37°C. for 2 hours to form a conjugate pad. The conjugate pad was thenlaminated onto the other end (closer to the detection zone) of theporous membrane. A cellulose wicking pad (Millipore Co.) sample pad wasfurther laminated onto the conjugate pad. The laminated full card wasthen cut into 4-millimeter wide lateral flow assay devices.

EXAMPLE 5

The ability to detect CRP in accordance with the present invention wasdemonstrated. Fifteen lateral flow devices (Samples 1-15) were made asdescribed in Example 4 and divided into three groups. The first groupwas stored at room temperature, the second group was heated at 65° C.for 40 minutes, and the third group was heated at 65° C. for 210minutes. A mixing solution containing 60 microliters of CRP (100nanograms per milliliter) and 60 microliters of β-hCG (50 nanograms permilliliter) in 20 millimolar Hepes buffer (pH of 7.51) with 1% Tween® 20(ICI Americas) was applied to the sample pad of the first group. Amixing solution containing 60 microliters of CRP (100 nanograms permilliliter) and 60 microliters of β-hCG (50 nanograms per milliliter) in20 millimolar Hepes buffer (pH of 8.0) with 1% Tween® 20 (ICI Americas)was applied to the sample pad of the second group. A mixing solutioncontaining 60 microliters of CRP (100 nanograms per milliliter) and 60microliters of β-hCG (50 nanograms per milliliter) in 20 millimolarHepes buffer (pH of 8.5) with 1% Tween® 20 (ICI Americas) was applied tothe sample pad of the third group.

After 30 minutes of development at room temperature, the color densityof the detection zone and calibration zone was measured using areflectance spectrophotometer. The statistical data for each group arelisted below in Table 5.

TABLE 5 Statistical Data Signal Standard Device pH Intensity MeanDeviation % CV 1-5 7.51 I_(d) 88.720 2.885 3.252 I_(d)/I_(c) 1.044400.00729 0.698  6-10 8.00 I_(d) 84.3080 1.5491 1.837 I_(d)/I_(c) 1.203400.00769 0.639 11-15 8.50 I_(d) 88.910 2.675 3.009 I_(d)/I_(c) 1.047150.00629 0.601

As indicated, the calibrated data for the detection zone had a muchlower standard deviation and % CV than the non-calibrated data over arange of pH values.

While the invention has been described in detail with respect to thespecific embodiments thereof, it will be appreciated that those skilledin the art, upon attaining an understanding of the foregoing, mayreadily conceive of alterations to, variations of, and equivalents tothese embodiments. Accordingly, the scope of the present inventionshould be assessed as that of the appended claims and any equivalentsthereto.

1. A diagnostic test kit for detecting the presence or quantity of a test analyte within a test sample, the diagnostic test kit comprising: a lateral flow assay device comprising a porous membrane, the porous membrane defining a detection zone and a calibration zone, wherein a first receptive material is immobilized within the detection zone and a second receptive material is immobilized within the calibration zone; a plurality of assay reagents, wherein the assay reagents comprise a calibration analyte that is disposed on the lateral flow device, wherein the calibration analyte and the test analyte are members of the same protein family, the assay reagents further comprising detection probes and calibration probes disposed on the lateral flow device prior to the application of a test sample to the device, the detection probes being disposed upstream from the detection zone on the lateral flow device, the calibration probes being disposed upstream from the calibration zone on the lateral flow device, the detection probes being conjugated with a first specific binding member configured to preferentially bind to the test analyte and the calibration probes being conjugated with a second specific binding member configured to preferentially bind to the calibration analyte, further wherein the first receptive material is configured to preferentially bind to the test analyte to form a sandwich complex, and the second receptive material is configured to preferentially bind to the calibration analyte to form a sandwich complex; and the detection zone being capable of producing a detection signal and the calibration zone being capable of producing a calibration signal, the intensity of the detection signal, as calibrated by the intensity of the calibration signal, being directly proportional to the concentration of the test analyte within the test sample.
 2. The diagnostic test kit of claim 1, wherein the detection probes comprise a luminescent compound or a visual label.
 3. The diagnostic test kit of claim 1, wherein the first specific binding member, the second specific binding member, the first receptive material, and the second receptive material are immunoreactive binding members.
 4. The diagnostic test kit of claim 1, wherein the test analyte is a pentraxin protein.
 5. The diagnostic test kit of claim 4, wherein the test analyte is C-reactive protein.
 6. The diagnostic test kit of claim 4, wherein the calibration analyte is a pentraxin protein.
 7. The diagnostic test kit of claim 6, wherein the calibration analyte is a phosphocholine-binding pentraxin protein.
 8. The diagnostic test kit of claim 7, wherein the calibration analyte is pentraxin 3, neuronal pentraxin 1, or neuronal pentraxin
 2. 9. The diagnostic test kit of claim 1, wherein the calibration zone is located downstream from the detection zone.
 10. The diagnostic test kit of claim 1, wherein the lateral flow assay device further comprises a conjugate pad in fluid communication with the porous membrane, wherein one or more of the assay reagents are disposed on the conjugate pad.
 11. The diagnostic test kit of claim 10, wherein the detection probes and calibration probes are disposed on the conjugate pad.
 12. The diagnostic test kit of claim 10, further comprising a sample pad that is positioned upstream from the conjugate pad and defines a point of application for the test sample, wherein the calibration analyte is disposed on the sample pad.
 13. The diagnostic test kit of claim 1, wherein one or more of the assay reagents are disposed on the lateral flow device at a location that is downstream from a point of application for the test sample.
 14. The diagnostic test kit of claim 1, wherein one or more of the assay reagents are disposed on the lateral flow device at a location that is upstream from a point of application for the test sample.
 15. A diagnostic test kit for detecting the presence or quantity of a test analyte within a test sample, the diagnostic test kit comprising: a lateral flow assay device comprising a porous membrane, the porous membrane defining a detection zone and a calibration zone, wherein a first receptive material is immobilized within the detection zone and a second receptive material is immobilized within the calibration zone; a calibration analyte disposed on the lateral flow assay device at a first area, wherein the calibration analyte and the test analyte are members of the same protein family; detection probes disposed on the lateral flow assay device, the detection probes being disposed upstream from the detection zone on the lateral flow device, the detection probes being conjugated with a first specific binding member configured to preferentially bind to the test analyte; and calibration probes disposed on the lateral flow assay device at a second area prior to the application of a test sample to the device, the calibration probes being disposed upstream from the calibration zone on the lateral flow device, the calibration probes being conjugated with a second specific binding member configured to preferentially bind to the calibration analyte, further wherein the first receptive material is configured to preferentially bind to the test analyte to form a sandwich complex, and the second receptive material is configured to preferentially bind to the calibration analyte to form a sandwich complex; and the detection zone being capable of producing a detection signal and the calibration zone being capable of producing a calibration signal, the intensity of the detection signal, as calibrated by the intensity of the calibration signal, being directly proportional to the concentration of the test analyte within the test sample.
 16. The diagnostic test kit of claim 15, wherein the detection probes comprise a luminescent compound or a visual label.
 17. The diagnostic test kit of claim 15, wherein the first specific binding member, the second specific binding member, the first receptive material, and the second receptive material are immunoreactive binding members.
 18. The diagnostic test kit of claim 15, wherein the test analyte is a pentraxin protein.
 19. The diagnostic test kit of claim 18, wherein the calibration analyte is a pentraxin protein.
 20. The diagnostic test kit of claim 15, wherein the calibration zone is located downstream from the detection zone.
 21. The diagnostic test kit of claim 15, wherein the first area is upstream of the second area.
 22. The diagnostic test kit of claim 15, wherein the detection probes are disposed in the second area.
 23. The diagnostic test kit of claim 15, wherein the first area is a sample pad and the second area is a conjugate pad. 